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J Appl Physiol 88: 1167-1174, 2000;
8750-7587/00 $5.00
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Vol. 88, Issue 4, 1167-1174, April 2000

Prostaglandins potentiate U-46619-induced pulmonary microvascular dysfunction

Joseph K. Wright1, Lawrence T. Kim1, Thomas E. Rogers2, and Richard H. Turnage1

Departments of 1 Surgery and 2 Pathology, University of Texas Southwestern Medical School and Dallas Veterans Affairs Medical Center, Dallas, Texas 75216


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The induction of cyclooxygenase is an important event in the pathophysiology of acute lung injury. The purpose of this study was to examine the synergistic effects of various cyclooxygenase products (PGE2, PGI2, PGF2alpha ) on thromboxane A2 (TxA2)-mediated pulmonary microvascular dysfunction. The lungs of Sprague-Dawley rats were perfused ex vivo with Krebs-Henseleit buffer containing indomethacin and PGE2 (5 × 10-8 to 1 × 10-7 M), PGF2alpha (7 × 10-9 to 5 × 10-6 M), or PGI2 (5 × 10-8 to 2 × 10-5 M). The TxA2-receptor agonist U-46619 (7 × 10-8 M) was then added to the perfusate, and then the capillary filtration coefficient (Kf), pulmonary arterial pressure (Ppa), and total pulmonary vascular resistance (RT) were determined. The Kf of lungs perfused with U-46619 was twice that of lungs perfused with buffer alone (P = 0.05). The presence of PGE2, PGF2alpha , and PGI2 within the perfusate of lungs exposed to U-46619 caused 118, 65, and 68% increases in Kf, respectively, over that of lungs perfused with U-46619 alone (P < 0.03). The RT of lungs perfused with PGE2 + U-46619 was ~30% greater than that of lungs exposed to either U-46619 (P < 0.02) or PGE2 (P < 0.01) alone. When paired measurements of RT taken before and then 15 min after the addition of U-46619 were compared, PGI2 was found to attenuate U-46619-induced increases in RT (P < 0.01). These data suggest that PGE2, PGI2, and PGF2alpha potentiate the effects of TxA2-receptor activation on pulmonary microvascular permeability.

capillary filtration coefficient; pulmonary vascular resistance; isolated perfused lung model


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THROMBOXANE A2 (TxA2) has been incriminated as an important mediator of the pulmonary microvascular dysfunction that characterizes tissue ischemia and reperfusion injury (29), endotoxin exposure (9), and cutaneous thermal injury (14). In these conditions, as well as others, TxA2 has been shown to cause vasoconstriction and enhanced microvascular permeability. Upregulation of cyclooxygenase is an important event in the generation of TxA2 during acute inflammatory states. Cyclooxygenase catalyzes the incorporation of molecular oxygen into arachidonic acid, yielding a cyclic endoperoxide (PGG2); subsequent peroxidation yields PGH2, the precursor for the synthesis of TxA2 and PGE2, PGI2, and PGF2alpha . In general, each of these cyclooxygenase products is released by the lung in response to a particular inflammatory stimulus, albeit in varying amounts (9, 14).

Despite their common origin, the physiological effects of these substances on the pulmonary microvasculature are diverse. For example, TxA2 and PGF2alpha are constrictors of the pulmonary vasculature in rats, whereas PGI2 is a potent vasodilator (4, 5). Furthermore, TxA2 profoundly increases microvascular permeability, whereas the other agents have little, if any, effect by themselves (20, 27). Several investigators have reported that PGE2 and PGI2 potentiate the effects of histamine, bradykinin, and interleukin-1 on microvascular permeability (2, 34, 35). This observation, as well as the frequency with which PGE2 and other prostaglandins are released with TxA2 during acute inflammatory states, led us to postulate that PGE2, PGI2, and PGF2alpha potentiate the proinflammatory effects of TxA2 on the pulmonary microvasculature.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolated, Perfused Lung Model

Pathogen-free Sprague-Dawley rats (250-350 g) were anesthetized with pentobarbital sodium (40 mg/kg ip). A median sternotomy was performed, and the pulmonary arterial trunk and left atrium were cannulated via the right and left ventricles, respectively. The heart and lungs were excised en bloc, and the lungs were suspended by a ligature from a force transducer (TSD 125C; Biopac Systems, Santa Barbara, CA) for continuous measurement of lung weight. The lungs were perfused with Krebs-Henseleit buffer containing 3% BSA at 0.04 ml · g body wt-1 · min-1 and ventilated with room air at a rate of 60 strokes per minute. The Krebs-Henseleit buffer consists of an aqueous solution containing (in mM) 128 NaCl, 4.7 KCl, 1.2 MgSO4, 3.2 CaCl2, 1.2 KH2PO4, 25 NaHCO3, and 6.7 dextrose. The perfusate was bubbled with a 95% O2-5% CO2 mixture to maintain a normal pH (7.40-7.5). Pulmonary arterial pressure (Ppa) and pulmonary venous pressure (Ppv) were continuously measured with pressure transducers (TSD 104A; Biopac Systems) with zero reference at the level of the apex of the lung. These measurements were continuously recorded by a Biopac data acquisition unit (MP100 manager version 3.2.3, hardware version 1.1f, Biopac Systems) interfaced with a personal computer (Dell Computer, Austin, TX). The perfusate was maintained in a 37°C water bath. The first 75 ml of perfusate were discarded to remove blood elements from the vascular space; thereafter, the perfusate was recirculated. The total volume of the recirculating buffer was 70 ml. In each case, the lungs were perfused under zone III conditions with arterial pressure > venous pressure > airway pressure. The mean airway pressure was 2-3 mmHg and was maintained below Ppv (3-4 mmHg) by altering the height of the venous reservoir. The lung preparations were isogravimetric immediately before all measurements of pulmonary vascular tone and permeability.

Experimental Protocol

Determination of the effect of indomethacin on U-46619-induced pulmonary microvascular dysfunction. An initial set of experiments (Fig. 1A) was performed to determine the effect of indomethacin on U-46619-induced changes in pulmonary microvascular function. There were four experimental groups defined by the composition of the perfusate: 1) Krebs-Henseleit buffer alone (n = 5), 2) Krebs-Henseleit buffer + U-46619 (n = 7), 3) Krebs-Henseleit buffer + indomethacin (n = 8), and 4) Krebs-Henseleit buffer + indomethacin + U-46619 (n = 6). In this study, indomethacin (100 µM; Sigma Chemical, St. Louis, MO) was added to the perfusate at the beginning of the experiment; 15 min later, U-46619 (7.1 × 10-8 M) was added to the perfusate of the appropriate experimental groups. After an additional 15 min of ex vivo perfusion, pulmonary microvascular dysfunction was assessed by measuring the capillary filtration coefficient (Kf), total vascular resistance (RT), and pulmonary vascular pressures as described below.




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  		Fig. 1.   A: experimental protocol study examining effect of indomethacin on U-46619-induced changes in pulmonary microvascular function. Lungs of normal Sprague-Dawley rats (n = 5-8 per experimental group) were harvested and perfused ex vivo with Krebs-Henseleit buffer with or without indomethacin (100 µM). Fifteen minutes later, baseline measurements of pulmonary arterial pressure (Ppa) and pulmonary venous pressure (Ppv) were obtained and U-46619 (7.1 × 10-8 M) was added to perfusate. Fifteen minutes later, Ppa, Ppv, and capillary pressure (Pc) were measured, and the capillary filtration coefficient (Kf) and total vascular resistance (RT) were calculated. B: experimental protocol for study examining effect of PGE2 and PGF2alpha on U-46619-induced changes in pulmonary microvascular function. Lungs of normal Sprague-Dawley rats (n = 4-8 per experimental group) were harvested and perfused ex vivo with Krebs-Henseleit buffer with 100 µM indomethacin with or without PGE2 (5 × 10-8 M to 1 × 10-7 M) or PGF2alpha (7 × 10-9 M to 6 × 10-6 M). Fifteen minutes later, baseline measurements of Ppa and Ppv were obtained, and U-46619 (7.1 × 10-8 M) was added to perfusate. Fifteen minutes later, Ppa, Ppv, and Pc were measured, and Kf and RT were calculated. C: experimental protocol for study examining effect of PGI2 on U-46619-induced changes in pulmonary microvascular function. Lungs of normal Sprague-Dawley rats (n = 4-8 per experimental group) were harvested and perfused ex vivo with Krebs-Henseleit buffer containing 100 µM indomethacin for 15 min. PGI2 (5 × 10-8 M to 2 × 10-5 M) was added to perfusate, and 1 min later baseline measurements for Ppa and Ppv were obtained. U-46619 (7.1 × 10-8 M) was then added to perfusate, and 15 min later Ppa, Ppv, and Pc were measured, and Kf and RT were calculated.

U-46619 (9,11-dideoxy-11alpha ,9alpha -epoxymethanoprostaglandin F2alpha ) is a thromboxane-endoperoxide receptor agonist that has been commonly used to mimic the physiological effects of TxA2 (4, 22, 23, 25). The concentration of U-46619 was chosen based on a dose-response curve relating increasing concentrations of U-46619 to increases in Kf in this experimental system (data not shown). This concentration of U-46619 is associated with increases in Kf similar to those seen with in vivo models of acute lung injury (29). Lastly, the increases in Kf associated with U-46619 have been found to be completely inhibited by the thromboxane receptor antagonist SQ-29548 (2 µM; data not shown).

Determination of the effect of PGE2 and PGF2alpha on U-46619-induced pulmonary microvascular dysfunction. In these and subsequent experiments (Fig. 1B), indomethacin (100 µM) was added to the perfusate of the isolated, perfused lung model to inhibit the generation of endogenous prostanoids (3, 5, 37, 38). This concentration has been previously shown to inhibit the release of TxA2, PGF2alpha , and PGI2 in an experimental model similar to that employed in these experiments (3, 5). In this particular study, PGE2 (Oxford Biomedical Research, Oxford, MI; 1 × 10-8 M, 5 × 10-8 M, 1 × 10-7 M) or PGF2alpha (Cayman Chemical, Ann Arbor, MI; 7 × 10-9 M, 5 × 10-7 M, 1 × 10-6 M, 5 × 10-6 M) was added to the perfusate at the beginning of the experiment; 15 min later, baseline measurements of Ppa and Ppv were obtained. U-46619 (7.1 × 10-8 M) was then added to the perfusate and allowed to circulate for 15 min, after which Ppa and Ppv were again measured and the Kf and vascular resistance were determined.

PGE2 and PGF2alpha were prepared in Krebs-Henseleit buffer according to the supplier's recommendations. The concentrations and mode of administration of PGE2 and PGF2alpha in these experiments were based on the experience of other investigators utilizing similar experimental models (4, 5, 15, 23, 30, 34). The addition of these substances to the Krebs-Henseleit buffer had no effect on the pH of the perfusate.

Determination of the effect of PGI2 on U-46619-induced pulmonary microvascular dysfunction. In this experiment (Fig. 1C), the ex vivo lung model was perfused with Krebs-Henseleit buffer containing indomethacin (100 µM) for 15 min as described above. PGI2 (Oxford Biomedical Research; 2 × 10-8 M, 5 × 10-8 M, 2 × 10-7 M, 2 × 10-6 M, 2 × 10-5 M) was then added to the perfusate, and afterward baseline measurements of Ppa and Ppv were obtained. Immediately thereafter, U-46619 (7.1 × 10-8 M) was added to the perfusate; 7 min later a second infusion of PGI2 was given as described above. Seven minutes later (15 min after the addition of U-46619), Ppa and Ppv were measured, and vascular resistance and Kf were determined.

The protocol for administering PGI2 was based on its short half-life at a physiological pH (2-3 min). It was prepared in 50 mM Tris buffer (pH 9.0), as recommended by the supplier, and added to the perfusate immediately following reconstitution. The bioactivity of this preparation was confirmed by its ability to inhibit U-46619-induced vasoconstriction (4). The addition of PGI2 to the Krebs-Henseleit buffer as described above had no effect on the pH of the perfusate.

Measurement of Pulmonary Microvascular Dysfunction

Kf. Pulmonary microvascular permeability was quantitated by determining Kf as has been previously described by our laboratory (29) and that of other investigators (11, 39). Briefly, 15 min after the addition of U-46619, the capillary pressure prior to elevating Ppv (Pcpre) was measured using the double occlusion technique (1, 28). Ppv was then elevated 8-10 mmHg by raising the height of the venous reservoir. This results in a two-component weight gain consisting of an initial rapid increase related primarily to recruitment and distension of the vascular bed (minutes 0-1) and a second slow constant weight increase due to fluid filtration across the microvasculature (minutes 1-5). After 5 min of elevated Ppv, Pc was again measured before returning Ppv to baseline (Pcpost). Kf was calculated as shown in Eq. 1
<IT>K</IT><SUB>f</SUB> = <FR><NU>&Dgr;W/&Dgr;T</NU><DE>&Dgr;P</DE></FR> (1)
where Delta W is the change in lung weight between minutes 1 and 5 of partial venous outflow occlusion, Delta T is the duration of elevated Ppv during which Delta W is measured, and Delta P is the difference between Pcpost and Pcpre. Kf is normalized to body weight (expressed as g · min-1 · mmHg-1 · 100 g body wt-1). This methodology correlates well with the time 0 extrapolation technique (39).

Pulmonary vascular resistance. Immediately before the addition of U-46619, Ppa and Ppv were recorded and compared with measurements obtained 15 min after the addition of U-46619. RT was calculated as the total pressure drop across the lung as expressed in Eq. 2
R<SC>t</SC> = <FR><NU>Ppa − Ppv</NU><DE><A><AC>Q</AC><AC>˙</AC></A></DE></FR> (2)
where Q is the flow through the isolated perfused lung. In the isogravimetric state, the pulmonary circulation can be represented as a simple linear model in which Ppa is separated from Pc by a precapillary resistance (arterial resistance, Ra) and Pc is separated from Ppv by a postcapillary resistance (venous resistance, Rv) (1). Therefore, where RT is determined in the isogravimetric state, Ra and Rv can be calculated as follows
R<SUB>a</SUB> = <FR><NU>Ppa − Pc</NU><DE><A><AC>Q</AC><AC>˙</AC></A></DE></FR> (3)

R<SUB>v</SUB> = <FR><NU>Pc − Ppv</NU><DE><A><AC>Q</AC><AC>˙</AC></A></DE></FR> (4)

R<SC>t</SC> = R<SUB>a</SUB> + R<SUB>v</SUB> (5)
All resistance calculations (RT, Ra, and Rv) were normalized for body weight (expressed as mmHg · ml-1 · min-1 · 100 g body wt-1). RT was also expressed as the absolute difference in determinations of RT obtained before and 15 min after the addition of U-46619 to the ex vivo lung perfusion. This methodology minimizes variability between lung perfusions in that each lung serves as its own control. Furthermore, this methodology allows determination of the effect of each PG on U-46619-induced vasomotor activity after quantitation of the PG's effect on basal vasomotor tone.

In these determinations of Kf and vascular resistance, Pc was measured with the double occlusion method as described by Allison et al. (1) and Townsley et al. (28). This methodology has been demonstrated to correlate closely with measurements of isogravimetric capillary pressure in both normal and acutely injured lungs (1, 28).

Statistical Analysis

All data are expressed as means ± SE. The sample sizes of the experimental groups ranged from four to eight. The data were compared by ANOVA with a Student-Newman-Keuls test (SigmaStat; Version 2.0; SPSS; Chicago, IL). Statistical significance was considered for a type 1 error of <5%. All P values represent the results of post hoc comparisons. All experiments were approved by the Committee on the Care and Use of Animals at the University of Texas Southwestern Medical School and Dallas Veterans Affairs Medical Center.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Indomethacin on Pulmonary Microvascular Function

The Kf of lungs exposed to U-46619 in the absence of indomethacin was more than three times greater than that of lungs perfused with Krebs-Henseleit buffer alone (Fig. 2; P < 0.02). The Kf of lungs exposed to U-46619 in the presence of indomethacin (100 µM) was about twice that of lungs perfused with Krebs-Henseleit buffer containing indomethacin but without U-46619 (P = 0.05). The Kf of lungs exposed to U-46619 in the presence of indomethacin was ~40% less than that of lungs perfused with U-46619 without indomethacin (P < 0.03). Lesser concentrations of indomethacin (10 µM) did not have this effect in that the Kf of lungs exposed to U-46619 in the presence of 10 µM indomethacin was 0.023 ± 0.005 g · min-1 · mmHg-1 · 100 g body wt-1; this value was not different from that of lungs exposed to U-46619 in the absence of indomethacin, which was 0.019 ± 0.003 g · min-1 · mmHg-1 · 100 g body wt-1. As shown in Fig. 2, indomethacin (100 µM) did not appear to affect the baseline permeability of the lungs.


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  		Fig. 2.   Effect of indomethacin on U-46619-induced changes in pulmonary Kf. Lungs of normal Sprague-Dawley rats were perfused ex vivo with Krebs-Henseleit buffer with or without indomethacin (100 µM). Fifteen minutes later, TxA2-receptor agonist U-46619 (7.1 × 10-8 M) was added to perfusate of half of these lungs. After an additional 15 min, pulmonary microvascular permeability was assessed by measuring Kf. * P < 0.05 vs. lungs perfused without U-46619; # P < 0.05 vs. lungs perfused with U-46619 but in absence of indomethacin.

Indomethacin also appeared to have important effects on U-46619-induced vasoconstriction. In the absence of indomethacin, U-46619-induced vasoconstriction was evidenced by a 22% (P = 0.04), 36% (P < 0.01), and 19% (P = 0.01) increase in Ppa, RT, and Pc, respectively, when compared with measurements in lungs perfused with Krebs-Henseleit buffer alone. These data are shown in Table 1. In the presence of indomethacin, U-46619 caused a 22% increase in Ppa (P < 0.01) but no significant change in Pc (P = 0.16) or RT (P = 0.1). Furthermore, the RT of lungs perfused with indomethacin + U-46619 was 25% less than that of lungs exposed to U-46619 in the absence of indomethacin (P = 0.01). Indomethacin's effect on U-46619-induced vasoconstriction appeared to be localized primarily to the precapillary location, in that the Ra of lungs exposed to indomethacin + U-46619 was ~30% less than that of lungs exposed to U-46619 in the absence of indomethacin (P = 0.01). Of interest, the presence of indomethacin (100 µM) within the perfusate did not appear to affect basal pulmonary vasomotor tone in the absence of U-46619.

                              
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  		Table 1.   Effect of indomethacin (100 µM) on U-46619-induced changes in pulmonary vasomotor tone

Effects of Prostaglandins on Pulmonary Microvascular Function

Kf. The presence of PGE2 (5 × 10-8 M), PGF2alpha (5 × 10-6 M), or PGI2 (5 × 10-8 M) within the perfusate significantly enhanced the effect of U-46619 on pulmonary microvascular permeability. These data are shown in Fig. 3. The Kf of lungs perfused with U-46619 + PGE2, PGF2alpha , or PGI2 was 118, 65, and 68% greater, respectively, than that of lungs perfused with U-46619 alone (P < 0.03). Furthermore, the Kf of lungs perfused with U-46619 + PGE2, PGF2alpha , or PGI2 was 4.4, 2.3, and 2.3 times that of lungs perfused with the respective prostaglandins alone (P < 0.01). In the absence of U-46619, PGE2, PGF2alpha , and PGI2, at the same concentrations as used above, had no effect on pulmonary microvascular permeability.


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  		Fig. 3.   Synergistic effect of PGE2, PGI2, and PGF2alpha on U-46619-induced increases in pulmonary Kf. Lungs of normal Sprague-Dawley rats were perfused ex vivo with Krebs-Henseleit buffer containing 100 µM indomethacin and PGE2, PGF2alpha , or PGI2. U-46619 (7.1 × 10-8 M) was then added to perfusate, and 15 min later Kf was determined. Solid bars represent lungs perfused with U-46619, whereas open bars represent lungs perfused in absence of U-46619 (means ± SE). * P < 0.01 vs. lungs perfused with Krebs-Henseleit buffer + indomethacin (Krebs alone); # P < 0.02 vs. lungs perfused with Krebs-Henseleit buffer + indomethacin + U-46619; Psi  P < 0.002 vs. lungs perfused with same prostaglandin but in absence of U-46619.

Concentrations of PGE2 <5 × 10-8 M had no significant effect on U-46619-induced increases in Kf, whereas 1 × 10-7 M was associated with an increase in Kf similar to that of 5 × 10-8 M. The Kf of lungs exposed to U-46619 and 1 × 10-8 M PGE2 was not different from that of lungs exposed to U-46619 alone (0.009 ± 0.0004 vs. 0.011 ± 0.001 g · min-1 · mmHg-1 · 100 g body wt-1; n = 4 and 7, respectively). In contrast, the Kf of lungs exposed to U-46619 and either 5 × 10-8 M PGE2 (0.024 ± 0.004 g · min-1 · mmHg-1 · 100 g body wt-1; n = 6) or 1 × 10-7 M PGE2 (0.0245 ± 0.004 g · min-1 · mmHg-1 · 100 g body wt-1; n = 4) was significantly greater than that of lungs perfused with U-46619 alone (0.011 ± 0.001 g · min-1 · mmHg-1 · 100 g body wt-1; n = 7; P < 0.05) or indomethacin alone (0.006 ± 0.001 g · min-1 · mmHg-1 · 100 g body wt-1; n = 8; P < 0.05). Concentrations of PGF2alpha <5 µM (7 × 10-9 to 1 × 10-6 M) and concentrations of PGI2 <5 × 10-8 (2 × 10-8 M) had no effect on U-46619-induced changes in Kf, whereas concentrations >5 × 10-8 M had an effect similar to the latter (data not shown).

Pulmonary Vascular Pressures and Resistance

The effect of PGE2 (5 × 10-8 M), PGF2alpha (5 × 10-6 M), and PGI2 (5 × 10-8 M) on pulmonary vasomotor tone is shown in Table 2. In the absence of U-46619, the addition of PGE2, PGF2alpha , or PGI2 to the perfusate of the ex vivo lung model had no effect on Ppa or Ppv when compared with those lungs perfused with buffer alone. Of interest, the RT of lungs perfused with PGF2alpha or PGI2 was significantly greater than that of lungs exposed to Krebs-Henseleit buffer alone (P = 0.01 for both substances). This appeared to be due principally to a 40% greater Ra in the lungs exposed to these prostaglandins (P = 0.01). The RT of lungs perfused with PGE2 was not statistically different from that of lungs perfused with buffer alone (P = 0.057).

                              
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  		Table 2.   Effect of PGE2 (5 × 10-8 M), PGF2alpha (5 × 10-6 M), and PGI2 (5 × 10-8 M) on U-46619-induced changes in pulmonary vasomotor tone

The RT of lungs exposed to PGE2 + U-46619 was significantly greater than that of lungs exposed to U-46619 alone (P < 0.02). This increase appeared to be due principally to a 49% increase in Ra over that of lungs exposed to U-46619 alone (P < 0.01). In contrast, PGE2 + U-46619 did not appear to alter either Ppa or Pc when compared with measurements taken in lungs exposed to U-46619 alone. The addition of U-46619 to lungs perfused with PGF2alpha or PGI2 did not increase Ppa, Pc, or RT over that associated with the prostaglandin itself or that caused by U-46619 alone.

Figure 4 illustrates the effect of PGE2, PGF2alpha , and PGI2 on U-46619-induced vasoconstriction by comparing the RT of the ex vivo lung model immediately before and 15 min after the addition of U-46619 to the perfusate. In these paired measurements, U-46619 caused a significant increase in RT (P < 0.05) compared with measurements taken immediately before the addition of U-46619. PGI2 attenuated the increase in RT associated with U-46619 (P < 0.01), whereas PGE2 and PGF2alpha had no significant effect. Expression of the Ppa data in this manner produced identical results (data not shown).


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  		Fig. 4.   Effect of PGE2, PGF2alpha , and PGI2 on U-46619-induced increases in RT. Lungs of normal Sprague-Dawley rats were perfused ex vivo with Krebs-Henseleit buffer containing 100 µM indomethacin and PGE2, PGF2alpha , or PGI2. Pulmonary arterial and venous pressure was measured before and 15 min after addition of U-46619 (7.1 × 10-8 M). These data are expressed as difference in measurements of RT before and 15 min after addition of U-46619 to perfusate. Solid bars represent of lungs perfused with U-46619, whereas open bars represent lungs perfused in absence of U-46619 (means ± SE). * P < 0.001 vs. Krebs-Henseleit buffer + indomethacin (Krebs-Henseleit alone); # P < 0.001 vs. lungs perfused with Krebs-Henseleit buffer + indomethacin + U-46619; Psi  P < 0.002 vs. lungs perfused with same prostaglandin but in absence of U-46619.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The generation of TxA2 by the lung during acute inflammatory states is often accompanied by the release of PGE2, PGF2alpha , and PGI2 (9, 14). Although synergism between PGE2 and PGI2 and various proinflammatory substances such as histamine, bradykinin, and interleukin-1 has been well described (2, 34, 35), there have been few studies examining the effect of these prostaglandins on TxA2-mediated changes in pulmonary microvascular permeability. The data presented in this study suggest the following: 1) PGE2, PGF2alpha , and PGI2 do not alter microvascular permeability when administered individually into an isolated, buffer-perfused rat lung; 2) each of these prostaglandins potentiates the proinflammatory effects of TxA2-receptor activation on pulmonary microvascular permeability; 3) indomethacin (100 µM) attenuates U-46619-induced effects on pulmonary microvascular permeability and vascular resistance; and 4) PGI2 attenuates TxA2-induced vasoconstriction.

Various investigators (12, 20, 21, 27) have examined the individual effects of PGE2, PGF2alpha , and PGI2 on pulmonary microvascular permeability in normal animal models, including the rat. In contradistinction to their very active role in the regulation of local vasomotor tone, the results of the present study, as well as those of other investigators (20, 21, 27), suggest that these exogenously administered prostaglandins do not directly alter pulmonary vascular permeability, at least when administered into a buffer-perfused lung model in the concentrations utilized in this study.

To the authors' knowledge, there are no studies examining the effect of PGE2 and PGF2alpha on TxA2-mediated changes in pulmonary microvascular permeability and only two studies examining the effect of PGI2 (37, 38). In the latter two reports (37, 38), the investigators, utilizing an ex vivo newborn lamb lung model, found that the Kf of lungs exposed to PGI2 and a TxA2-receptor agonist (9,11-epithio-11,12-methano-TxA2) was significantly greater than that of lungs exposed to either PGI2 or TxA2-receptor activation alone. This increase in fluid filtration was accompanied by a reduction in vascular hydrostatic pressure, suggesting that, in addition to a direct effect on microvascular permeability, PGI2 may have increased pulmonary Kf by increasing vascular surface area (37, 38). These results are similar to those of the present study in that the Kf of lungs exposed to PGI2 and U-46619 was nearly 70% greater than that of lungs exposed to U-46619 alone (P < 0.02). Furthermore, this occurred in a setting in which PGI2 prevented U-46619-induced vasoconstriction. Of interest in one of the Yoshimura studies (38), PGI2 alone was found to induce an acute lung injury manifested by "a diffuse hemorrhagic edema." This is clearly different from the experience reported in the present study [as well as that reported by other investigators (12, 21)] in which PGI2 alone had no effect on Kf. Even in the presence of U-46619, the increase in Kf associated with PGI2 exposure was much more modest than that suggested by Yoshimura et al. (38). The reason for this difference is unclear, although fundamental differences in the experimental models are likely to be important [e.g., a sanguineous perfusate was utilized in the Yoshimura study (38), whereas an asanguineous buffer was used in the present study]. Furthermore, the total amount of PGI2 administered in the Yoshimura study was more than 100 times greater than that of the present study (about 300 µg over 180 min in the Yoshimura study vs. 2.6 µg in the present study).

It is also possible that neutrophils, sequestered within the ex vivo lung model during harvesting, may have contributed to the enhanced microvascular function that characterized exposure of the lungs to U-46619 + PGE2 or PGF2alpha . The importance of sequestered neutrophils in the microvascular dysfunction of the ex vivo perfused lung model was initially suggested by Seibert et al. (24) in 1993 in a study in which neutrophils sequestered within the perfused lung were found to contribute significantly to the enhanced permeability associated with pulmonary ischemia-reperfusion injury. One may postulate that in the present study perfusion of the lung with U-46619 [and perhaps PGF2alpha and PGI2 (13, 17, 33, 40)] may have acted on sequestered neutrophils, resulting in the generation of a respiratory burst and, ultimately, a neutrophil-mediated microvascular dysfunction (18, 26). Other more recent studies have suggested that U-46619, as well as PGE2, PGF2alpha , and PGI2, inhibits neutrophil activation, at least as manifested by increases in intracellular free calcium, leukoaggregation, and the release of superoxide radical, beta -glucuronidase, and leukotriene B4 (22, 32, 36). These more recent observations would appear to be inconsistent with the notion that U-46619 or PGE2, PGF2alpha , or PGI2 increased pulmonary microvascular permeability by activating on neutrophils sequestered within the lung.

Other studies have suggested that activation of the endothelial cell TxA2/PGH2 receptor may directly alter microvascular permeability by changing the endothelial cytoskeletal structure, with resultant changes in cell shape and cell-cell continuity (31). This would suggest a possible second mechanism by which PGF2alpha and PGI2 may work through the TxA2/PGH2 receptor to enhance microvascular permeability (13, 17, 33, 40). Although these studies clearly suggest that PGF2alpha and PGI2 may activate the TxA2/PGH2 receptor, the relative affinity of TxA2 receptors for PGI2 and PGF2alpha is extremely low when compared with that of TxA2 for U-46619 (19, 33, 40). Furthermore, most of these studies were performed using pharmacological doses in in vitro models. Lastly, PGE2 has been shown to promote bradykinin-induced edema formation (within the skin), although the mechanism by which this occurs (i.e., whether it is related to neutrophil activation via the EP3 receptor, a direct effect on the microvascular permeability, or an effect of increased blood flow) remains speculative (2, 6, 34, 35). Furthermore, to the investigators' knowledge, no one has examined the effect of circulating PGE2 on pulmonary vasomotor tone or permeability in a model similar to that utilized in the present study.

In contrast to the paucity of studies examining the effects of PGE2, PGF2alpha , and PGI2 on TxA2-mediated changes in pulmonary microvascular permeability, there have been numerous studies examining the potential therapeutic effects of prostaglandins, particularly PGE2 and PGI2, on pulmonary microvascular dysfunction (7, 10). Brigham et al. (7) have shown that the administration of PGE2 limits endotoxin-induced increases in pulmonary microvascular permeability. In a more recent study, the induction of PGE2 and PGI2 release by transfecting a recombinant cyclooxygenase gene into the pulmonary microvasculature was found to attenuate endotoxin-induced vasoconstriction and pulmonary edema (8), an effect attributed, at least in part, to PGE2- and PGI2-mediated inhibition of TxA2 release by the lung itself or inflammatory cells such as neutrophils or platelets sequestered within the lung (8). If a similar mechanism had been operative in the present study, one would have anticipated a reduction, not increase, in Kf. Furthermore, the concentration of TxB2 (the stable metabolite of TxA2) was measured within the perfusate of several of the lungs exposed to PGE2, PGI2, and PGF2alpha and was found to be no different from that of lungs not exposed to these prostaglandins (data not shown).

The addition of indomethacin to the perfusate of the ex vivo lung model appeared to attenuate, but not prevent, U-46619-induced increases in Kf. Previous investigators have suggested that indomethacin attenuates increases in pulmonary microvascular permeability by inhibiting the release of proinflammatory prostanoids, particularly TxA2, by the lung (16, 39). In the present study, the addition of U-46619 to the perfusate resulted in a small but statistically significant increase in the release of PGE2 and TxA2 by the lung. The presence of 100 µM indomethacin within the perfusate prevented this increase in endogenous PGE2 and TxA2 release (data not shown), suggesting that the "protective" effect of indomethacin resulted from an inhibition of endogenous prostaglandin and/or TxA2 release.

In the present study, the RT of lungs perfused with PGI2 was 40% greater than that of lungs perfused with buffer alone (P = 0.007), whereas PGI2 totally prevented the increase in vasoconstriction associated with U-46619 exposure. These results are consistent with those of Zhao et al. (40) and Williams et al. (33), who demonstrated that higher concentrations of PGI2 contract rat pulmonary artery rings or aortic strips [probably via the TxA2/PGH2 receptor (33, 40)], whereas lower concentrations and those given in the presence of vasoconstrictors [e.g., U-46619 (4) or PGF2alpha (5)] cause vasodilation [possibly via the PGI2/PGE1 receptor (33)]. The results of the present study are consistent with the observations of other investigators who suggested that PGI2 becomes a more potent vasodilator as the tone of the blood vessel is increased (5).

Similar to PGI2, the RT of lungs perfused with PGF2alpha was significantly greater than that of lungs perfused with Krebs-Henseleit buffer (P = 0.01), an increase due principally to vasoconstriction of the precapillary segment. This observation is nearly identical to that published by Barnard et al. (5) using a similar experimental model. In contrast to PGI2, however, PGF2alpha had no effect on U-46619-induced vasoconstriction. This is perhaps related to the fact that PGF2alpha competes less effectively for the TxA2/PGH2 receptor than does U-46619 (5, 17, 25).

There is very little information available regarding the effect of PGE2 on pulmonary vasomotor function. In the present study, the RT of lungs perfused with U-46619 + PGE2 was significantly greater than that of either U-46619 (P < 0.02) or PGE2 alone (P < 0.05), suggesting that PGE2 potentiated the vasoconstriction associated with U-46619. Enthusiasm for this conclusion is limited by the observations that 1) the Ppa of lungs exposed to PGE2 + U-46619 was not different from that of lungs exposed to U-46619 alone and 2) the presence of PGE2 within the perfusate of the lungs did not appear to affect U-46619-induced vasoconstriction when assessed as the absolute change in RT and Ppa in paired measurements taken immediately before and 15 min after the addition of U-46619 (as shown in Fig. 5).

In summary, these data are consistent with the hypothesis that PGE2, PGI2, and PGF2alpha potentiate the proinflammatory effects of TxA2-receptor activation on pulmonary microvascular permeability. Although the increase in pulmonary Kf due to PGI2 + U-46619 may result, in part, from PGI2-induced increases in vascular surface area, there is little evidence to support such a mechanism for the increase in Kf due to the combined effects of U-46619 + PGE2 and PGF2alpha . The synergistic effects of these prostaglandins on TxA2-induced pulmonary microvascular dysfunction may contribute to the lung injury that commonly accompanies systemic inflammatory states.


    ACKNOWLEDGEMENTS

This work is supported in part by a Department of Veterans Affairs Merit Review Grant and a Texas Chapter of the American Lung Association Research Grant.


    FOOTNOTES

This work was presented in part at the 21st Annual Conference on Shock, June 14-17, 1998, San Antonio, TX.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. H. Turnage, Dept. of Surgery, Univ. of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235.

Received 11 December 1998; accepted in final form 2 November 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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